Blood vessels are the means by which oxygen and nutrients are supplied to living tissues and waste products are removed from living tissue. Angiogenesis refers to the process by which new blood vessels are formed. See, for example, the review by Folkman and Shing, J. Biol. Chem. 267 (16), 10931-10934 (1992). Thus, where appropriate, angiogenesis is a critical biological process. It is essential in reproduction, development and wound repair. However, inappropriate angiogenesis can have severe negative consequences. For example, it is only after many solid tumors are vascularized as a result of angiogenesis that the tumors have a sufficient supply of oxygen and nutrients that permit it to grow rapidly and metastasize. Because maintaining the rate of angiogenesis in its proper equilibrium is so critical to a range of functions, it must be carefully regulated in order to maintain health. The angiogenesis process is believed to begin with the degradation of the basement membrane by proteases secreted from endothelial cells (EC) activated by mitogens such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). The cells migrate and proliferate, leading to the formation of solid endothelial cell sprouts into the stromal space, then, vascular loops are formed and capillary tubes develop with formation of tight junctions and deposition of new basement membrane.
In adults, the proliferation rate of endothelial cells is typically low compared to other cell types in the body. The turnover time of these cells can exceed one thousand days. Physiological exceptions in which angiogenesis results in rapid proliferation typically occurs under tight regulation, such as found in the female reproduction system and during wound healing.
The rate of angiogenesis involves a change in the local equilibrium between positive and negative regulators of the growth of microvessels. The therapeutic implications of angiogenic growth factors were first described by Folkman and colleagues over two decades ago (Folkman, N. Engl. J. Med., 285:1182-1186 (1971)). Abnormal angiogenesis occurs when the body loses at least some control of angiogenesis, resulting in either excessive or insufficient blood vessel growth. For instance, conditions such as ulcers, strokes, and heart attacks may result from the absence of angiogenesis normally required for natural healing. In contrast, excessive blood vessel proliferation can result in tumor growth, tumor spread, blindness, psoriasis and rheumatoid arthritis.
There are instances where a greater degree of angiogenesis is desirable--increasing blood circulation, wound healing, and ulcer healing. For example, recent investigations have established the feasibility of using recombinant angiogenic growth factors, such as fibroblast growth factor (FGF) family (Yanagisawa-Miwa, et al., Science, 257:1401-1403 (1992) and Baffour, et al., J Vasc Surg, 16:181-91 (1992)), endothelial cell growth factor (ECGF) (Pu, et al., J Surg Res, 54:575-83 (1993)), and more recently, vascular endothelial growth factor (VEGF) to expedite and/or augment collateral artery development in animal models of myocardial and hindlimb ischemia (Takeshita, et al., Circulation, 90:228-234 (1994) and Takeshita, et al., J Clin Invest, 93:662-70 (1994)).
Conversely, there are instances, where inhibition of angiogenesis is desirable. For example, many diseases are driven by persistent unregulated angiogenesis, also sometimes referred to as "neovascularization." In arthritis, new capillary blood vessels invade the joint and destroy cartilage. In diabetes, new capillaries invade the vitreous, bleed, and cause blindness. Ocular neovascularization is the most common cause of blindness. Tumor growth and metastasis are angiogenesis-dependent. A tumor must continuously stimulate the growth of new capillary blood vessels for the tumor itself to grow.
The current approved treatment of these diseases is inadequate. Agents which prevent continued angiogenesis, e.g, drugs (TNP-470), monoclonal antibodies, antisense nucleic acids and proteins (angiostatin and endostatin) are currently being tested, but have not been approved. See, Battegay, J. Mol. Med., 73, 333-346 (1995); Hanahan et al., Cell, 86, 353-364 (1996); Folkman, N. Engl. J. Med., 333, 1757-1763 (1995). Although preliminary results with the antiangiogenic proteins are promising, they are relatively large in size and are difficult to use and produce. Moreover, proteins are subject to enzymatic degradation. Thus, new agents that inhibit angiogenesis are needed. New antiangeogenic proteins or peptides that show improvement in size, ease of production, stability and/or potency would be desirable.
Members of the FGF family are characterized by their high affinity for glycosaminoglycan and heparin, and their high mitogenicity for mesodermand neuroectoderm derived cells. Furthermore, they are among the most potent inducers of neovascularization (see Kan, M. et al., "An Essential Heparin-binding Domain in the Fibroblast Growth Factor Receptor Kinase", Science, Volume 259, (Mar. 26, 1993) pp. 1918-1921; Ornitz, D. M. et al. "Heparin is Required for Cell-free Binding of basic Fibroblast Growth Factor to a Soluble Receptor and for Mitogenesis in Whole Cells", Molecular and Cellular Biology, Volume 12, (January 1992) pp. 240-247; Klagsbrun, M. et al. "MINIREVIEW: A Dual Receptor System is Required for Basic Fibroblast Growth Factor Activity", Cell, pp. 229-231; Risau, W., "Angiogenic Growth Factors", Progress in Growth Factor Research, Volume 2, (1990) pp. 71-79; Bouck, N., "Tumor Angiogenesis: The Role of Oncogenes and Tumor Suppressor Genes", Cancer Cells, Volume 2, Number 6, (June 1990) pp. 179-185).
Several inhibitors of FGF-2 mitogenic activity have been described, including the synthetic polymers sulfated beta-cyclodextrins, sulfated malto-oligosaccharides and phophororothioate oligodeoxynucleotides as well as the drug suramin (Guimond, et al., Biol. Chem. 268, 23906-23914, Venkataraman, et al., Proc. Natl. Acad. Sci., USA 93, 845-850). While these inhibitors have been proposed to be anti-angiogenic agents, potentially useful in cancer chemotherapy and in preventing restenosis following vascular injury, their use as FGF-2 antagonists has been limited because of their anticoagulant potency or in vivo toxicity.
Thus, there is a need for FGF antagonists having low toxicity and anticoagulant activity.